The isolation and separation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein or peptide-based pharmaceutical compounds. Similarly there is also a need to separate biomolecules for analytical purposes in order to be able to quantify and identify the proteins and/or peptides present in a sample. Electrophoretic methods are commonly used in the separation step. A wide variety of methods are used for the detection and quantification of the separated proteins and/or peptides. For identification and characterisation of separated proteins mass spectrometry (MS) methods are normally used as these methods are fast and require very small amounts of proteins and/or peptides.
In isoelectric focusing (IEF), the separation takes place in a pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways: with the aid of a solution of carrier ampholytes or with an immobilised pH gradient.
In the case of an immobilised pH gradient (IPG) the charged or chargeable groups generating the pH gradient are bound either to the wall of a capillary system or to the matrix when some kind of gel is used to get convection stabilisation. The immobilised charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated. The concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode. A commercially available example of a system for generation of immobilised pH gradients is the IMMOBILINE II SYSTEM™ (Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed. Immobilised pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilised gradient determines the resulting pH gradient, while the carrier ampholytes contribute with conductivity.
A common problem with isoelectric focusing of proteins and/or peptides is that the focused proteins/peptides are unevenly distributed in the gel with bad resolution.
To solve this problem for proteins, a non-linear pH-gradient has been immobilised in IEF gels, for example IMMOBILINE DRYSTRIPS™ pH 3-11 NL (Amersham Biosciences, Uppsala, Sweden). This non-linear gradient cannot be used for focusing of peptides.
JP 1100448 A describes arranging several gel media in parallel relative to the direction of migration of proteins in isoelectric focusing. The gel media each comprise a different pH-range. Thus, this invention does not describe a continuous gel having a step-wise pH-gradient in the same gel. Furthermore, it does not describe focusing of peptides.
Thus, there is a need of improved gels or strips for isoelectric focusing of peptides with better resolution and distribution of the peptides after focusing.